Systematic engineering enables efficient biosynthesis of L-phenylalanine in E. coli from inexpensive aromatic precursors.

BACKGROUND: L-phenylalanine is an essential amino acid with various promising applications. The microbial pathway for L-phenylalanine synthesis from glucose in wild strains involves lengthy steps and stringent feedback regulation that limits the production yield. It is attractive to find other candidates, which could be used to establish a succinct and cost-effective pathway for L-phenylalanine production. Here, we developed an artificial bioconversion process to synthesize L-phenylalanine from inexpensive aromatic precursors (benzaldehyde or benzyl alcohol). In particular, this work opens the possibility of L-phenylalanine production from benzyl alcohol in a cofactor self-sufficient system without any addition of reductant. RESULTS: The engineered L-phenylalanine biosynthesis pathway comprises two modules: in the first module, aromatic precursors and glycine were converted into phenylpyruvate, the key precursor for L-phenylalanine. The highly active enzyme combination was natural threonine aldolase LtaEP.p and threonine dehydratase A8HB.t, which could produce phenylpyruvate in a titer of 4.3 g/L. Overexpression of gene ridA could further increase phenylpyruvate production by 16.3%, reaching up to 5 g/L. The second module catalyzed phenylpyruvate to L-phenylalanine, and the conversion rate of phenylpyruvate was up to 93% by co-expressing PheDH and FDHV120S. Then, the engineered E. coli containing these two modules could produce L-phenylalanine from benzaldehyde with a conversion rate of 69%. Finally, we expanded the aromatic precursors to produce L-phenylalanine from benzyl alcohol, and firstly constructed the cofactor self-sufficient biosynthetic pathway to synthesize L-phenylalanine without any additional reductant such as formate. CONCLUSION: Systematical bioconversion processes have been designed and constructed, which could provide a potential bio-based strategy for the production of high-value L-phenylalanine from low-cost starting materials aromatic precursors.

Engineering acetylation platform for the total biosynthesis of D-amino acids.

Optically pure D-amino acids are key chemicals with various applications. Although the production of specific D-amino acids has been achieved by chemical synthesis or with in vitro enzyme catalysts, it is challenging to convert a simple carbon source into D-amino acids with high efficiency. Here, we design an artificial metabolic pathway by engineering bacteria to heterologously express racemase and N-acetyltransferase to produce N-acetyl-D-amino acids from L-amino acids. This new platform allows the cytotoxicity of D-amino acids to be avoided. The universal potential of this acetylation protection strategy for effectively synthesizing optically pure D-amino acids is demonstrated by testing sixteen amino acid targets. Furthermore, we combine pathway optimization and metabolic engineering in Escherichia coli and achieve practically useful efficiency with four specific examples, including N-acetyl-D-valine, N-acetyl-D-serine, N-acetyl-D-phenylalanine and N-acetyl-D-phenylglycine, with titers reaching 5.65 g/L, 5.25 g/L, 8.025 g/L and 130 mg/L, respectively. This work opens up opportunities for synthesizing D-amino acids directly from simple carbon sources, avoiding costly and unsustainable conventional approaches.

Characteristics of genotype, drug resistance, and molecular transmission network among newly diagnosed HIV-1 infections in Shenzhen, China.

The HIV-1 pandemic has persisted for four decades, and poses a major challenge to global public health. Shenzhen, a city with large number of migrant populations in China, is suffering HIV-1 epidemic. It is necessary to continuously conduct the molecular surveillance among newly diagnosed HIV-1 patients in these migrant population. In this study, plasma samples of newly diagnosed and ART-naive HIV-1 infections were collected from Shenzhen city in China. The partial genes of HIV-1 gag and pol were amplified and sequenced for the analysis of genotype, drug resistance, and molecular transmission network. Ninety-one sequences of pol gene were obtained from newly diagnosed HIV-1 infections in Shenzhen, and seven HIV-1 subtypes were revealed in this investigation. Among them, the circulating recombinant form (CRF) 07_BC was the mostly frequent subtype (53.8%, 49/91), followed by CRF01_AE (20.9%, 19/91), CRF55_01B (9.9%, 9/91), unique recombinant forms (URFs) (8.8%, 8/91), B (3.3%, 3/91), CRF59_01B (2.2%, 2/91), and CRF08_BC (1.1%, 1/91). The overall prevalence of pretreatment drug resistance (PDR) was 23.1% (21/91), and 52.38% (11/21) of the PDR was specific for the nonnucleotide reverse transcriptase inhibitors (NNRTIs). Furthermore, a total of 3091 pol gene sequences were used to generate 19 molecular transmission clusters, and then one growing cluster, a new cluster, and a cluster with growth reactivation were identified. The result revealed that more sexual partner, CRF_07BC subtype, and seven amino acid deletions in gag p6 region might be the influencing factors associated with the high risk of transmission behavior. Compared with CRF01_AE subtype, CRF07_BC subtype strains were more likely to form clusters in molecular transmission network. This suggests that long-term surveillance of the HIV-1 molecular transmission should be a critical measure to achieve a precise intervention for controlling the spread of HIV-1 in China.

A novel strategy for L-arginine production in engineered Escherichia coli.

BACKGROUND: L-arginine is an important amino acid with applications in diverse industrial and pharmaceutical fields. N-acetylglutamate, synthesized from L-glutamate and acetyl-CoA, is a precursor of the L-arginine biosynthetic branch in microorganisms. The enzyme that produces N-acetylglutamate, N-acetylglutamate synthase, is allosterically inhibited by L-arginine. L-glutamate, as a central metabolite, provides carbon backbone for diverse biological compounds besides L-arginine. When glucose is the sole carbon source, the theoretical maximum carbon yield towards L-arginine is 96.7%, but the experimental highest yield was 51%. The gap of L-arginine yield indicates the regulation complexity of carbon flux and energy during the L-arginine biosynthesis. Besides endogenous biosynthesis, N-acetylglutamate, the key precursor of L-arginine, can be obtained by chemical acylation of L-glutamate with a high yield of 98%. To achieve high-yield production of L-arginine, we demonstrated a novel approach by directly feeding precursor N-acetylglutamate to engineered Escherichia coli. RESULTS: We reported a new approach for the high yield of L-arginine production in E. coli. Gene argA encoding N-acetylglutamate synthase was deleted to disable endogenous biosynthesis of N-acetylglutamate. The feasibility of external N-acetylglutamate towards L-arginine was verified via growth assay in argA- strain. To improve L-arginine production, astA encoding arginine N-succinyltransferase, speF encoding ornithine decarboxylase, speB encoding agmatinase, and argR encoding an arginine responsive repressor protein were disrupted. Based on overexpression of argDGI, argCBH operons, encoding enzymes of the L-arginine biosynthetic pathway, ~ 4 g/L L-arginine was produced in shake flask fermentation, resulting in a yield of 0.99 mol L-arginine/mol N-acetylglutamate. This strain was further engineered for the co-production of L-arginine and pyruvate by removing genes adhE, ldhA, poxB, pflB, and aceE, encoding enzymes involved in the conversion and degradation of pyruvate. The resulting strain was shown to produce 4 g/L L-arginine and 11.3 g/L pyruvate in shake flask fermentation. CONCLUSIONS: Here, we developed a novel approach to avoid the strict regulation of L-arginine on ArgA and overcome the metabolism complexity in the L-arginine biosynthesis pathway. We achieve a high yield of L-arginine production from N-acetylglutamate in E. coli. Co-production pyruvate and L-arginine was used as an example to increase the utilization of input carbon sources.

The information-motivation-behavioral skills (IMB) model of antiretroviral therapy (ART) adherence among people living with HIV in Shanghai.

The Information-Motivation-Behavioral skills (IMB) model of antiretroviral therapy (ART) adherence was applied in people living with HIV/AIDS in Shanghai, China to understand how adherence-related information, motivation and behavioral skills would affect ART adherence. The LifeWindows Information-Motivation-Behavioral Skills ART Adherence Questionnaire (LW-IMB-AAQ) was translated into Chinese and used. The IMB model was then implemented by testing standardized path estimates with standard model fitness indices in the participants. 426 participants from 11 community centres in Putuo district of Shanghai were recruited, of which 95.3% reported a high level of adherence (>95% adherence). The fitness indices of the final adjusted model were χ2 = 6.110, df = 7, p = 0.527(>0.05), CFI = 1.000(>0.9) and RMSEA = 0.000 (<0.08). In the model, information, which was separated into two sections (the perceived effect of ART on health and knowledge about ART medication), had an indirect effect on the ART adherence through behavioral skills, while motivation did not have such an effect. Neither information nor motivation had a direct effect on ART adherence. In addition, motivation was related to the two sections of information. The feasibility of the IMB model of ART adherence is verified by its application to predictive of adherence-related behaviors among HIV+ patients in this study.

Relationships of work stress and interpersonal needs with industrial workers' mental health: a moderated mediation model.

OBJECTIVES: This study explores whether feelings of defeat (i.e., a sense of failed struggle and losing rank; referred to as defeat for simplicity) mediated the effect of work stress on depression/anxiety, the effect of interpersonal needs on depression/anxiety for Chinese industrial workers, and the possible moderating role of social support. METHOD: A cross-sectional study was conducted in Shenzhen, China in 2019, in total, 2023 industrial workers (of 2700 invited; response rate = 75%) completed a self-administered survey consisted of Job Stress Scale, Interpersonal Needs Questionnaire, Defeat Scale, Centre for Epidemiological Studies Depression Scale, Generalized Anxiety Disorder Scale, two face-valid questions for social support, as well as sociodemographic information. Moderated mediation model was tested and loop plots were applied to probe into the conditional effects of work and interpersonal stress on depression and anxiety symptoms. RESULT: Both the direct and indirect effect of work stress on depression and anxiety through defeat were significant (Work stress→ Depression: B = 0.035, p < .001, Work stress→ Defeat→ Depression: B = 0.034, p < .001; Work stress→ Anxiety: B = 0.038, p < .001, Work stress→ Defeat→ Anxiety: B = 0.045, p < .001). Meanwhile, defeat mediated the relationship of interpersonal needs with depression partially and the relationship of interpersonal needs with anxiety totally (Interpersonal needs→ Anxiety: B = 0.133, p < .001, Interpersonal needs→ Defeat→ Anxiety: B = 0.010, p = .537). Social support moderated the indirect path between interpersonal needs and depression/anxiety and buffered the effect. CONCLUSION: The mediating role of defeat and the moderator role of social support in the relationship between stress and depression/anxiety were confirmed in industrial workers. Workers who reported more work and interpersonal stress would report more defeat feelings, and then exhibited more depression and anxiety symptoms; this mediation effect was stronger for those who had lower social support, respectively.

Sexual risk behaviours among factory workers in Shenzhen, China: a cross-sectional study.

BACKGROUND: Factory workers are a key population for HIV transmission in China, as they often engage in sexual risk behaviours. This study aims to evaluate sexual risk behaviours and associated factors among factory workers in Shenzhen, China. METHODS: A cross-sectional study was conducted by using multi-stage stratified cluster random sampling. Full-time workers aged ≥18years were eligible to participate in the study. A self-administered questionnaire was used to collect information. Univariate and multivariable logistic regression were applied to assess factors associated with sexual risk behaviours. RESULTS: A total of 2029 factory workers were included. Mean age was 37.2 (±4.4)years; 48.5% were men. Two-thirds (64.9%) had had vaginal intercourse. Their sexual risk behaviours included condomless sex with casual partners in the last sex episode (23.6%), multiple sex partners (11.5%) and engaging in commercial sex (8.4%), in the past year. Having HIV/AIDS knowledge (adjusted odds ratio (AOR) 0.41, 95% confidence interval (CI) 0.24-0.70) and using a condom at sexual debut (AOR 0.08, 95% CI 0.05-0.13) were factors associated with condomless sex with casual partners in the last sex episode. Males (AOR 3.03, 95% CI 1.96-4.69 and AOR 2.19, 95% CI 1.33-3.60), local workers (AOR 2.11, 95% CI 1.01-4.42 and AOR 3.42, 95% CI 1.63-7.21), being single (AOR 2.04, 95% CI 1.39-3.01 and AOR 2.49, 95% CI 1.61-3.87), having sexual debut aged Conclusions : Sexual risk behaviours were prevalent despite most participants having basic HIV/AIDS knowledge. Future workplace-based prevention programs should target factory workers and there should be a focus on enhanced sexual education to reduce HIV transmission in China.

A model to predict adherence to antiretroviral therapy among people living with HIV.

OBJECTIVES: Suboptimal adherence to antiretroviral therapy (ART) dramatically hampers the achievement of the UNAIDS HIV treatment targets. This study aimed to develop a theory-informed predictive model for ART adherence based on data from Chinese. METHODS: A cross-sectional study was conducted in Shenzhen, China, in December 2020. Participants were recruited through snowball sampling, completing a survey that included sociodemographic characteristics, HIV clinical information, Information-Motivation-Behavioural Skills (IMB) constructs and adherence to ART. CD4 counts and HIV viral load were extracted from medical records. A model to predict ART adherence was developed from a multivariable logistic regression with significant predictors selected by Least Absolute Shrinkage and Selection Operator (LASSO) regression. To evaluate the performance of the model, we tested the discriminatory capacity using the concordance index (C-index) and calibration accuracy using the Hosmer and Lemeshow test. RESULTS: The average age of the 651 people living with HIV (PLHIV) in the training group was 34.1±8.4 years, with 20.1% reporting suboptimal adherence. The mean age of the 276 PLHIV in the validation group was 33.9±8.2 years, and the prevalence of poor adherence was 22.1%. The suboptimal adherence model incorporates five predictors: education level, alcohol use, side effects, objective abilities and self-efficacy. Constructed by those predictors, the model showed a C-index of 0.739 (95% CI 0.703 to 0.772) in internal validation, which was confirmed be 0.717 via bootstrapping validation and remained modest in temporal validation (C-index 0.676). The calibration capacity was acceptable both in the training and in the validation groups (p>0.05). CONCLUSIONS: Our model accurately estimates ART adherence behaviours. The prediction tool can help identify individuals at greater risk for poor adherence and guide tailored interventions to optimise adherence.

Acceptability of male circumcision for HIV prevention among men who have sex with men in China: a short report.

Recent evidence shows that circumcision is associated with lower HIV prevalence among MSM. We assessed the acceptability of circumcision for preventing HIV and that of Shang Ring circumcision (SRC) among men who have sex with men (MSM) in China. 538 adult MSM were recruited from six cities in China between January and March 2019. Participants were surveyed by an online, self-administered questionnaire. The acceptability of circumcision was assessed before and after the potential protective effect of circumcision against HIV was informed, and subsequently men's willingness to undergo SRC was assessed. The level of circumcision was 16.4%. Of 450 uncircumcised MSM, their willingness to be circumcised in the following six months increased significantly from 32.2% to 55.6% after the information session. Three quarters of men who were willing to undergo circumcision accepted SRC. MSM who perceived that circumcision could maintain genital hygiene were more likely to accept circumcision after the information session, whereas those who regarded circumcision as an embarrassing surgery were disinclined to be circumcised. The low circumcision rate, along with its high acceptability in Chinese MSM, suggests a great potential benefit of circumcision intervention if proved effective. SRC might be a popular circumcision procedure in this population.

The association between entrapment and depression among migrant workers in China: a social rank theory based study.

BACKGROUND: Migrant workers are a group susceptible for depression evolution due to occupational maladaptive triggers. The social rank theory illustrates the pathology process from defensive adaptation to depression, pointing out the early prevention of depression by discovering entrapment. This study aims to reveal the relationship between migrant workers' entrapment and depressive symptoms. METHODS: A total of 1805 migrant workers in Shenzhen were recruited by stratified multi-stage sampling. Sample's demographic, behavioral and psychosocial characteristics were described and analyzed to reveal the relationship between entrapment and depressive symptoms. The Receiver Operator Characteristic was performed to find the optimal cut-off point of Entrapment Scale for predicting depressive symptoms. RESULTS: In the binary logistic regression of sociodemographic variables, migrant workers who were married (univariate odds ratio (ORu) = 0.69, 95% Confidence Interval (CI) = 0.56-0.84), owned 1 or 2 children (ORu = 0.71, 95% CI = 0.58-0.86), had been working over 10 years (ORu = 0.71, 95% CI = 0.54-0.95), earned > 4999 yuan per month (ORu = 0.68, 95% CI = 0.47-0.99; multivariate odds ratio (ORm) = 0.57, 95% CI = 0.38-0.87) or with low risks of alcohol use disorders (ORu = 0.51, 95% CI = 0.34-0.75) had lower risks of depressive symptoms. After adjusted the aforementioned significant sociodemographic variables, migrant workers with severer entrapment were more likely to have depressive symptoms (adjusted odds ratio (ORa) = 1.13, 95% CI = 1.12-1.15). Besides, the study proved the reliability and validity of the Chinese version Entrapment Scale, preferring a two-dimensional structure, and 11 was the optimal cut-off value of this scale for predicting depressive symptoms among migrant workers. CONCLUSIONS: This result indicates the potential value of entrapment according to the social rank theory on facilitating early prevention of migrant works' depression and the application value of Entrapment Scale for effectively measuring mental status among migrant workers.

Awareness and uptake of non-occupational post-exposure prophylaxis (nPEP) among gay, bisexual and other men who have sex with men in China: a cross-sectional study.

Non-occupational post-exposure prophylaxis (nPEP) is recommended to reduce the likelihood of HIV infection after potential exposure. However, little is known about this practice among Chinese gay, bisexual and other men who have sex with men (GBM). GBM were enrolled from both centers for disease prevention and control (CDCs) and community health centers in six cities in China. Multivariable logistics regression was used to assess factors associated with awareness of and willingness to use nPEP. A total of 516 eligible GBM were included, 67.2% of whom were aware of nPEP, 76.0% were willing to use nPEP, and 2.3% had ever used nPEP. GBM who had college or higher education, had disclosed sexual orientation, had increased number of male sex partners in the last 6 months, and had ever tested for HIV were more likely to be aware of nPEP. Willingness to use nPEP was significantly associated with college or higher education, STI history, gay app use, and awareness of nPEP prior to study. Uptake of nPEP is still low among MSM in China. Efforts are needed to improve awareness of and access to nPEP among GBM, especially those at higher risk of HIV infection.

Efficacy of HIV interventions among factory workers in low- and middle-income countries: a systematic review.

BACKGROUND: Factory workers in low- and middle-income countries (LMICs) are vulnerable to HIV transmission. Interventions are needed to prevent HIV in this population. We systematically reviewed published literature on the efficacy of various HIV interventions in reducing stigma, risk behaviors and HIV transmission among factory workers. METHODS: A systematic review was performed using predefined inclusion and exclusion criteria. Four databases (PubMed, PsycINFO, Scopus and EMBASE) were searched for relevant publications between January 1, 1990 and December 31, 2018. Two independent reviewers assessed the methodological quality of studies. RESULTS: Thirteen articles were included, with 2 randomized controlled trials and 11 cohort studies. Five interventions and their combinations were summarized. Educational intervention increased condom use and reduced the use of recreational drugs and alcohol before sex. Community intervention that proactively provide HIV counselling and testing (HCT) services could increase the detection rate of HIV and other sexually transmitted diseases (STDs). Lottery intervention increased HCT uptake and decreased HIV public stigma. Education combined with community intervention reduced the proportion of workers with casual sex and enhanced HIV knowledge. Peer education combined with community intervention increased the proportion of workers who were willing to take their partners to HCT. Policy intervention combined with peer education enhanced HIV knowledge, perceived condom accessibility and condom use with regular partners. CONCLUSIONS: Various interventions improved HIV knowledge, decreased HIV stigma and reduced HIV-related risk behaviors among factory workers in LMICs. The combination of multiple interventions tended to achieve better efficacy than a single intervention. Persistent combination interventions are essential to address HIV in this population.

Rectal douching among men who have sex with men in six cities in China: a cross-sectional study.

Background Rectal douching, which is a common practice among men who have sex men (MSM) as a preparation for anal sex, may potentially increase the risk of HIV infection. However, little is known about the practice of rectal douching among Chinese MSM. This study investigated the characteristics of rectal douching and factors associated with rectal douching. METHODS: Between January and March 2019, MSM aged ≥18 years in six cities in China were recruited to the study. Participants were asked to complete a self-administered online questionnaire. Chi-squared tests and multivariable logistic regression were used to examine factors associated with rectal douching by calculating odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: Of 485 MSM in this study, 278 (57.3%) practiced rectal douching in the preceding 6 months. The most common reasons for douching before and after anal sex were hygiene/cleanliness (78.6% and 66.1% respectively) and HIV/STI prevention (8.5% and 17.4% respectively). The most used enemas and equipment before and after anal sex were tap water (73.5% and 70.2% respectively) and showerhead hoses (76.1% and 75.2% respectively). Men who engaged in both insertive and receptive anal sex (OR 8.84; 95% CI 4.52-17.30), exclusively receptive anal sex (OR 6.56; 95% CI 3.55-12.13), condom-less anal intercourse (OR 1.64; 95% CI 1.03-2.63) and anal sex after alcohol use (OR 1.54; 95% CI 1.00-2.35) were more likely to perform douching. CONCLUSION: Rectal douching is common in MSM, some of whom may not realise the potential risks associated with douching. The co-occurrence of douching and other high-risk behaviours would make MSM more vulnerable to HIV. Health education aimed at informing MSM of the potential risks and addressing the high prevalence of douching are urgently needed.

Association between rectal douching and HIV and other sexually transmitted infections among men who have sex with men: a systematic review and meta-analysis.

BACKGROUND: Men who have sex with men (MSM) are disproportionately affected by HIV and other STIs worldwide. Rectal douching, which is commonly used by MSM in preparation for anal sex, may increase the risk of HIV and other STIs by injuring the rectal mucosa. Results from individual studies reporting associations between rectal douching and HIV and other STIs among MSM are inconsistent. We performed a systematic review and meta-analysis to estimate the association between rectal douching and HIV and other STIs among MSM. METHODS: We searched PubMed, Embase, Scopus and Web of Science for studies published from January 1970 to November 2018. Studies that reported ORs and 95% CIs of associations between rectal douching and infection with HIV/STIs, or reported enough data to calculate these estimates, were included. We assessed risk of bias using the Newcastle-Ottawa Scale. ORs were pooled using a random effects model. RESULTS: Twenty-eight eligible studies were identified in our review, of which 24 (20 398 participants) were included in the meta-analysis. Rectal douching was associated with increased odds of infection with HIV (OR 2.80, 95% CI 2.32 to 3.39), and any STI other than HIV (including hepatitis B virus (HBV), hepatitis C virus (HCV), chlamydia, gonorrhoea, syphilis and human papillomavirus) (OR 2.46, 95% CI 1.95 to 3.11) among MSM. For specific STIs, douching was associated with increased odds of viral hepatitis (HBV, HCV) (OR 3.29, 95% CI 2.79 to 3.87), and chlamydia or gonorrhoea (OR 3.25, 95% CI 2.02 to 5.23). These associations remained significant in studies that adjusted for potential confounders. CONCLUSION: Rectal douching may put MSM at increased risk for infection with HIV and other STIs. Longitudinal studies are needed to clarify this association, and health education materials should inform men of the potential for increased risk of infection with rectal douching.

Engineering the production of dipicolinic acid in E. coli.

Dicarboxylic acids, such as the phthalic acids and their derivatives, are monomeric components in several important polyesters and polyamides. In most cases, these compounds are derived from fossil fuels and are not easily biodegradable. Dipicolinic acid (DPA) is a biologically derived aromatic di-acid that has a similar structure to isophthalic acid. Furthermore, DPA has been shown to give rise to polyesters, is readily biodegradable, and is non-toxic. DPA is naturally produced by Bacillus and Clostridium species during sporulation and can comprise up to 15% of the dry weight of bacterial spores. In this paper we demonstrate the first heterologous production of DPA and identify the genes appropriate for gram-scale production in the industrial workhorse organism, E. coli. Initially, several combinations of genes from the lysine pathway, including lysC, asd, dapA, and dapB, were overexpressed to determine which genes are necessary for recombinant production in E. coli. The in vitro activity of dipicolinate synthase was then compared between Bacillus subtilis and Clostridium perfringens. Next, in order to improve DPA production from glucose, an optimized strain was created that lacked several genes (lysA, tdh, and metA), resulting in 5.21 g/L DPA when 5 g/L of aspartate was supplied. Then, several aspartate kinases and dipicolinate synthases were screened for optimal activity in E. coli. The optimal genes were combined with the overexpression of phosphoenolpyruvate carboxylase to develop a full biosynthetic pathway capable of producing a titer of 4.7 g/L DPA directly from glucose. In summary, we have performed a detailed biochemical study of the key pathway enzyme dipicolinate synthase and achieved scalable heterogeneous production of DPA in the workhorse organism E. coli.

Engineering nonphosphorylative metabolism to generate lignocellulose-derived products.

Conversion of lignocellulosic biomass into value-added products provides important environmental and economic benefits. Here we report the engineering of an unconventional metabolism for the production of tricarboxylic acid (TCA)-cycle derivatives from D-xylose, L-arabinose and D-galacturonate. We designed a growth-based selection platform to identify several gene clusters functional in Escherichia coli that can perform this nonphosphorylative assimilation of sugars into the TCA cycle in less than six steps. To demonstrate the application of this new metabolic platform, we built artificial biosynthetic pathways to 1,4-butanediol (BDO) with a theoretical molar yield of 100%. By screening and engineering downstream pathway enzymes, 2-ketoacid decarboxylases and alcohol dehydrogenases, we constructed E. coli strains capable of producing BDO from D-xylose, L-arabinose and D-galacturonate. The titers, rates and yields were higher than those previously reported using conventional pathways. This work demonstrates the potential of nonphosphorylative metabolism for biomanufacturing with improved biosynthetic efficiencies.

Rerouting carbon flux for optimized biosynthesis of mesaconate in Escherichia coli.

Mesaconate, a branched unsaturated dicarboxylic acid, has drawn great interest because of its versatile applications. In this work, we optimized the fermentation efficiency of Escherichia coli to produce mesaconate from glucose. We first drove the carbon flux to 2-ketoglutarate by overexpressing genes involved in TCA precursor pathway and anaplerotic pathways. Then, to increase the pool of phosphoenolpyruvate (PEP), an upstream precursor for 2-ketoglutarate, the phosphotransferase system (PTS) of E. coli was inactivated by deleting glucose PTS permease and the import of glucose was altered by overexpressing galactose/H+ symporter GalP. Further, production optimization was achieved by deleting a class I fumarase (FumA) to block the hydration of mesaconate. Finally, we overexpressed PEP synthase (PpsA) to increase the availability of phosphoenolpyruvate and accelerate the production of mesaconate. These genetic modifications led to mesaconate production with a titer of 23.1 g L-1 and a yield of 0.46 g g-1 glucose (64% of the theoretical maximum). This work demonstrates the possibility of engineering a highly efficient bacteria strain that converts glucose into mesaconate with promising titer, rate, and yield.

Scalable production of mechanically tunable block polymers from sugar.

Development of sustainable and biodegradable materials is essential for future growth of the chemical industry. For a renewable product to be commercially competitive, it must be economically viable on an industrial scale and possess properties akin or superior to existing petroleum-derived analogs. Few biobased polymers have met this formidable challenge. To address this challenge, we describe an efficient biobased route to the branched lactone, ß-methyl-δ-valerolactone (ßMδVL), which can be transformed into a rubbery (i.e., low glass transition temperature) polymer. We further demonstrate that block copolymerization of ßMδVL and lactide leads to a new class of high-performance polyesters with tunable mechanical properties. Key features of this work include the creation of a total biosynthetic route to produce ßMδVL, an efficient semisynthetic approach that employs high-yielding chemical reactions to transform mevalonate to ßMδVL, and the use of controlled polymerization techniques to produce well-defined PLA-PßMδVL-PLA triblock polymers, where PLA stands for poly(lactide). This comprehensive strategy offers an economically viable approach to sustainable plastics and elastomers for a broad range of applications.