Carbon dioxide valorization into resveratrol via lithoautotrophic fermentation using engineered Cupriavidus necator H16.

BACKGROUND: Industrial biomanufacturing of value-added products using CO2 as a carbon source is considered more sustainable, cost-effective and resource-efficient than using common carbohydrate feedstocks. Cupriavidus necator H16 is a representative H2-oxidizing lithoautotrophic bacterium that can be utilized to valorize CO2 into valuable chemicals and has recently gained much attention as a promising platform host for versatile C1-based biomanufacturing. Since this microbial platform is genetically tractable and has a high-flux carbon storage pathway, it has been engineered to produce a variety of valuable compounds from renewable carbon sources. In this study, the bacterium was engineered to produce resveratrol autotrophically using an artificial phenylpropanoid pathway. RESULTS: The heterologous genes involved in the resveratrol biosynthetic pathway-tyrosine ammonia lyase (TAL), 4-coumaroyl CoA ligase (4CL), and stilbene synthase (STS) -were implemented in C. necator H16. The overexpression of acetyl-CoA carboxylase (ACC), disruption of the PHB synthetic pathway, and an increase in the copy number of STS genes enhanced resveratrol production. In particular, the increased copies of VvSTS derived from Vitis vinifera resulted a 2-fold improvement in resveratrol synthesis from fructose. The final engineered CR-5 strain produced 1.9 mg/L of resveratrol from CO2 and tyrosine via lithoautotrophic fermentation. CONCLUSIONS: To the best of our knowledge, this study is the first to describe the valorization of CO2 into polyphenolic compounds by engineering a phenylpropanoid pathway using the lithoautotrophic bacterium C. necator H16, demonstrating the potential of this strain a platform for sustainable chemical production.

Biotechnological Plastic Degradation and Valorization Using Systems Metabolic Engineering.

Various kinds of plastics have been developed over the past century, vastly improving the quality of life. However, the indiscriminate production and irresponsible management of plastics have led to the accumulation of plastic waste, emerging as a pressing environmental concern. To establish a clean and sustainable plastic economy, plastic recycling becomes imperative to mitigate resource depletion and replace non-eco-friendly processes, such as incineration. Although chemical and mechanical recycling technologies exist, the prevalence of composite plastics in product manufacturing complicates recycling efforts. In recent years, the biodegradation of plastics using enzymes and microorganisms has been reported, opening a new possibility for biotechnological plastic degradation and bio-upcycling. This review provides an overview of microbial strains capable of degrading various plastics, highlighting key enzymes and their role. In addition, recent advances in plastic waste valorization technology based on systems metabolic engineering are explored in detail. Finally, future perspectives on systems metabolic engineering strategies to develop a circular plastic bioeconomy are discussed.

Engineering Cupriavidus necator H16 for enhanced lithoautotrophic poly(3-hydroxybutyrate) production from CO2.

BACKGROUND: A representative hydrogen-oxidizing bacterium Cupriavidus necator H16 has attracted much attention as hosts to recycle carbon dioxide (CO2) into a biodegradable polymer, poly(R)-3-hydroxybutyrate (PHB). Although C. necator H16 has been used as a model PHB producer, the PHB production rate from CO2 is still too low for commercialization. RESULTS: Here, we engineer the carbon fixation metabolism to improve CO2 utilization and increase PHB production. We explore the possibilities to enhance the lithoautotrophic cell growth and PHB production by introducing additional copies of transcriptional regulators involved in Calvin Benson Bassham (CBB) cycle. Both cbbR and regA-overexpressing strains showed the positive phenotypes for 11% increased biomass accumulation and 28% increased PHB production. The transcriptional changes of key genes involved in CO2-fixing metabolism and PHB production were investigated. CONCLUSIONS: The global transcriptional regulator RegA plays an important role in the regulation of carbon fixation and shows the possibility to improve autotrophic cell growth and PHB accumulation by increasing its expression level. This work represents another step forward in better understanding and improving the lithoautotrophic PHB production by C. necator H16.

Improving Lipid Production of Yarrowia lipolytica by the Aldehyde Dehydrogenase-Mediated Furfural Detoxification.

Yarrowia lipolytica, the non-conventional yeast capable of high lipogenesis, is a microbial chassis for producing lipid-based biofuels and chemicals from renewable resources such as lignocellulosic biomass. However, the low tolerance of Y. lipolytica against furfural, a major inhibitory furan aldehyde derived from the pretreatment processes of lignocellulosic biomass, has restricted the efficient conversion of lignocellulosic hydrolysates. In this study, the furfural tolerance of Y. lipolytica has been improved by supporting its endogenous detoxification mechanism. Specifically, the endogenous genes encoding the aldehyde dehydrogenase family proteins were overexpressed in Y. lipolytica to support the conversion of furfural to furoic acid. Among them, YALI0E15400p (FALDH2) has shown the highest conversion rate of furfural to furoic acid and resulted in two-fold increased cell growth and lipid production in the presence of 0.4 g/L of furfural. To our knowledge, this is the first report to identify the native furfural detoxification mechanism and increase furfural resistance through rational engineering in Y. lipolytica. Overall, these results will improve the potential of Y. lipolytica to produce lipids and other value-added chemicals from a carbon-neutral feedstock of lignocellulosic biomass.

Complete Genome Sequence of Paenibacillus sp. CAA11: A Promising Microbial Host for Lignocellulosic Biorefinery with Consolidated Processing.

Several bioprocessing technologies, such as separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), and consolidated bioprocessing (CBP), have been highlighted to produce bio-based fuels and chemicals from lignocellulosic biomass. Successful CBP, an efficient and economical lignocellulosic biorefinery process compared with other processes, requires microorganisms with sufficient cellulolytic activity and biofuel/chemical-producing ability. Here, we report the complete genome of Paenibacillus sp. CAA11, a newly isolated promising microbial host for CBP-producing ethanol and organic acids from cellulose. The genome of Paenibacillus sp. CAA11 comprises one 4,888,410 bp chromosome with a G + C content of 48.68% containing 4418 protein-coding genes, 102 tRNA genes, and 39 rRNA genes. The functionally active cellulase, encoded by CAA_GH5 was identified to belong to glycosyl hydrolase family 5 (GH5) and consisted of a catalytic domain and a cellulose-binding domain 3 (CBM3). When cellulolytic activity of CAA_GH5 was assayed through Congo red method by measuring the size of halo zone, the recombinant Bacillus subtilis RIK1285 expressing CAA_GH5 showed a comparable cellulolytic activity to B. subtilis RIK1285 expressing Cel5, a previously verified powerful bacterial cellulase. This study demonstrates the potential of Paenibacillus sp. CAA11 as a CBP-enabling microbe for cost-effective biofuels/chemicals production from lignocellulosic biomass.

RNA-guided single/double gene repressions in Corynebacterium glutamicum using an efficient CRISPR interference and its application to industrial strain.

BACKGROUND: The construction of microbial cell factories requires cost-effective and rapid strain development through metabolic engineering. Recently, RNA-guided CRISPR technologies have been developed for metabolic engineering of industrially-relevant host. RESULTS: To demonstrate the application of the CRISPR interference (CRISPRi), we developed two-plasmid CRISPRi vectors and applied the CRISPRi in Corynebacterium glutamicum to repress single target genes and double target genes simultaneously. Four-different single genes (the pyc, gltA, idsA, and glgC genes) repressions were successfully performed using the CRISPRi vectors, resulting significant mRNA reductions of the targets compared to a control. Subsequently, the phenotypes for the target gene-repressed strains were analyzed, showing the expected cell growth behaviors with different carbon sources. In addition, double gene repression (the idsA and glgC genes in a different order) by the CRISPRi resulted in an independent gene repression to each target gene simultaneously. To demonstrate an industrial application of the CRISPRi, citrate synthase (CS)-targeting DM1919 (L-lysine producer) strains with a sgRNA-gltA-r showed reduced CS activity, resulting in the improvement of L-lysine yield by 1.39-fold than the parental DM1919 (a lysine producer). CONCLUSIONS: Single or double gene repression were successfully performed using the CRISPRi vectors and sequence specific sgRNAs. The CRISPRi can be applied for multiplex metabolic engineering to enhanced lysine production and it will promote the further rapid development of microbial cell factories of C. glutamicum.

High production of 2,3-butanediol from glycerol without 1,3-propanediol formation by Raoultella ornithinolytica B6.

Conversion of crude glycerol derived from biodiesel processes to value-added chemicals has attracted much attention. Herein, Raoultella ornithinolytica B6 was investigated for the high production of 2,3-butanediol (2,3-BD) from glycerol without 1,3-propanediol (1,3-PD) formation, a by-product hindering 2,3-BD purification. By evaluating the effects of temperature, agitation speed, and pH control strategy, the fermentation conditions favoring 2,3-BD production were found to be 25 °C, 400 rpm, and pH control with a lower limit of 5.5, respectively. Notably, significant pH fluctuations which positively affect 2,3-BD production were generated by simply controlling the lower pH limit at 5.5. In fed-batch fermentation under those conditions, R. ornithinolytica B6 produced 2,3-BD up to 79.25 g/L, and further enhancement of 2,3-BD production (89.45 g/L) was achieved by overexpressing homologous 2,3-BD synthesis genes (the budABC). When pretreated crude glycerol was used as a sole carbon source, R. ornithinolytica B6 overexpressing budABC produced 78.10 g/L of 2,3-BD with the yield of 0.42 g/g and the productivity of 0.62 g/L/h. The 2,3-BD titer, yield, and productivity values obtained in this study are the highest 2,3-BD production from glycerol among 1,3-PD synthesis-deficient 2,3-BD producers, demonstrating R. ornithinolytica B6 as a promising 2,3-BD producer from glycerol.

Engineering of a modular and synthetic phosphoketolase pathway for photosynthetic production of acetone from CO2 in Synechococcus elongatus PCC 7942 under light and aerobic condition.

Capture and conversion of CO2 to valuable chemicals is intended to answer global challenges on environmental issues, climate change and energy security. Engineered cyanobacteria have been enabled to produce industry-relevant chemicals from CO2 . However, the final products from cyanobacteria have often been mixed with fermented metabolites during dark fermentation. In this study, our engineering of Synechococcus elongatus PCC 7942 enabled continuous conversion of CO2 to volatile acetone as sole product. This process occurred during lighted, aerobic culture via both ATP-driven malonyl-CoA synthesis pathway and heterologous phosphoketolase (PHK)-phosphotransacetylase (Pta) pathway. Because of strong correlations between the metabolic pathways of acetate and acetone, supplying the acetyl-CoA directly from CO2 in the engineered strain, led to sole production of acetone (22.48 mg/L ± 1.00) without changing nutritional constraints, and without an anaerobic shift. Our engineered S. elongatus strains, designed for acetone production, could be modified to create biosolar cell factories for sustainable photosynthetic production of acetyl-CoA-derived biochemicals.

Burkholderia jirisanensis sp. nov., isolated from forest soil.

A Gram-negative, catalase-positive, mesophilic, obligately aerobic bacterium designated JRM2-1T was isolated from forest soil of Jirisan Mountain, Republic of Korea, and its taxonomic position was investigated based on a polyphasic taxonomic approach. Cells of strain JRM2-1T grew optimally at pH 5.0-7.0 and at 25 °C. Strain JRM2-1T was susceptible to chloramphenicol, gentamicin, kanamycin, nalidixic acid, rifampicin, streptomycin and tetracycline. On the basis of 16S rRNA gene sequence similarity, the closest neighbour of strain JRM2-1T was Burkholderia rhizosphaerae WR43T (98.1 %). On the basis of our phylogenetic analysis, strain JRM2-1T is clearly distinguished from related species of the genus Burkholderia and is clustered with plant-associated members of the genus. The major cellular fatty acids were C16 : 0, C17 : 0 cyclo and C19 : 0 cyclo ω8c. The polar lipid profile of strain JRM2-1T contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, several unidentified aminolipids and an unidentified aminophospholipid. The isoprenoid quinone of strain JRM2-1T was Q-8 and the DNA G+C content was 63.7 mol%. On the basis of our polyphasic taxonomic investigation, strain JRM2-1T is considered to represent a novel species in the genus Burkholderia, for which the name Burkholderia jirisanensis sp. nov. is proposed. The type strain is JRM2-1T ( = AIM 0373T = KCTC 42072T = JCM 19985T).

Adaptive evolution and metabolic engineering of a cellobiose- and xylose- negative Corynebacterium glutamicum that co-utilizes cellobiose and xylose.

BACKGROUND: An efficient microbial cell factory requires a microorganism that can utilize a broad range of substrates to economically produce value-added chemicals and fuels. The industrially important bacterium Corynebacterium glutamicum has been studied to broaden substrate utilizations for lignocellulose-derived sugars. However, C. glutamicum ATCC 13032 is incapable of PTS-dependent utilization of cellobiose because it has missing genes annotated to ß-glucosidases (bG) and cellobiose-specific PTS permease. RESULTS: We have engineered and evolved a cellobiose-negative and xylose-negative C. glutamicum that utilizes cellobiose as sole carbon and co-ferments cellobiose and xylose. NGS-genomic and DNA microarray-transcriptomic analysis revealed the multiple genetic mutations for the evolved cellobiose-utilizing strains. As a result, a consortium of mutated transporters and metabolic and auxiliary proteins was responsible for the efficient cellobiose uptake. Evolved and engineered strains expressing an intracellular bG showed a better rate of growth rate on cellobiose as sole carbon source than did other bG-secreting or bG-displaying C. glutamicum strains under aerobic culture. Our strain was also capable of co-fermenting cellobiose and xylose without a biphasic growth, although additional pentose transporter expression did not enhance the xylose uptake rate. We subsequently assessed the strains for simultaneous saccharification and fermentation of cellulosic substrates derived from Canadian Ponderosa Pine. CONCLUSIONS: The combinatorial strategies of metabolic engineering and adaptive evolution enabled to construct C. glutamicum strains that were able to co-ferment cellobiose and xylose. This work could be useful in development of recombinant C. glutamicum strains for efficient lignocellulosic-biomass conversion to produce value-added chemicals and fuels.

Biomass, strain engineering, and fermentation processes for butanol production by solventogenic clostridia.

Butanol is considered an attractive biofuel and a commercially important bulk chemical. However, economical production of butanol by solventogenic clostridia, e.g., via fermentative production of acetone-butanol-ethanol (ABE), is hampered by low fermentation performance, mainly as a result of toxicity of butanol to microorganisms and high substrate costs. Recently, sugars from marine macroalgae and syngas were recognized as potent carbon sources in biomass feedstocks that are abundant and do not compete for arable land with edible crops. With the aid of systems metabolic engineering, many researchers have developed clostridial strains with improved performance on fermentation of these substrates. Alternatively, fermentation strategies integrated with butanol recovery processes such as adsorption, gas stripping, liquid-liquid extraction, and pervaporation have been designed to increase the overall titer of butanol and volumetric productivity. Nevertheless, for economically feasible production of butanol, innovative strategies based on recent research should be implemented. This review describes and discusses recent advances in the development of biomass feedstocks, microbial strains, and fermentation processes for butanol production.

Deletion of the budBAC operon in Klebsiella pneumoniae to understand the physiological role of 2,3-butanediol biosynthesis.

Klebsiella pneumoniae is known to produce 2,3-butanediol (2,3-BDO), a valuable chemical. In K. pneumoniae, the 2,3-BDO operon (budBAC) is involved in the production of 2,3-BDO. To observe the physiological role of the 2,3-BDO operon in a mixed acid fermentation, we constructed a budBAC-deleted strain (SGSB109). The production of extracellular metabolites, CO2 emission, carbon distribution, and NADH/NAD(+) balance of SGSB109 were compared with the parent strain (SGSB100). When comparing the carbon distribution at 15 hr, four significant differences were observed: in 2,3-BDO biosynthesis, lactate and acetate production (lactate and acetate production increased 2.3-fold and 4.1-fold in SGSB109 compared to SGSB100), CO2 emission (higher in SGSB100), and carbon substrate uptake (higher in SGSB100). Previous studies on the inactivation of the 2,3-BDO operon were focused on the increase of 1,3-propanediol production. Few studies have been done observing the role of 2,3-BDO biosynthesis. This study provides a prime insight into the role of 2,3-BDO biosynthesis of K. pneumoniae.

Caproiciproducens galactitolivorans gen. nov., sp. nov., a bacterium capable of producing caproic acid from galactitol, isolated from a wastewater treatment plant.

A strictly anaerobic, Gram-stain-positive, non-spore-forming, rod-shaped bacterial strain, designated BS-1T, was isolated from an anaerobic digestion reactor during a study of bacteria utilizing galactitol as the carbon source. Its cells were 0.3-0.5 µm × 2-4 µm, and they grew at 35-45 °C and at pH 6.0-8.0. Strain BS-1T produced H2, CO2, ethanol, acetic acid, butyric acid and caproic acid as metabolic end products of anaerobic fermentation. Phylogenetic analysis, based on the 16S rRNA gene sequence, showed that strain BS-1T represented a novel bacterial genus within the family Ruminococcaceae, Clostridium Cluster IV. The type strains that were most closely related to strain BS-1T were Clostridium sporosphaeroides KCTC 5598T (94.5 %), Clostridium leptum KCTC 5155T (94.3 %), Ruminococcus bromii ATCC 27255T (92.1 %) and Ethanoligenens harbinense YUAN-3T (91.9 %). Strain BS-1T had 17.6 % and 20.9 % DNA-DNA relatedness values with C. sporosphaeroides DSM 1294T and C. leptum DSM 753T, respectively. The major components of the cellular fatty acids were C16 : 0 dimethyl aldehyde (DMA) (22.1 %), C16 : 0 aldehyde (14.1 %) and summed feature 11 (iso-C17 : 0 3-OH and/or C18 : 2 DMA; 10.0 %). The genomic DNA G+C content was 50.0 mol%. Phenotypic and phylogenetic characteristics allowed strain BS-1T to be clearly distinguished from other taxa of the genus Clostridium Cluster IV. On the basis of these data, the isolate is considered to represent a novel genus and novel species within Clostridium Cluster IV, for which the name Caproiciproducens galactitolivorans gen. nov., sp. nov. is proposed. The type species is BS-1T ( = JCM 30532T and KCCM 43048T).

Compounds inhibiting the bioconversion of hydrothermally pretreated lignocellulose.

Hydrothermal pretreatment using liquid hot water, steam explosion, or dilute acids enhances the enzymatic digestibility of cellulose by altering the chemical and/or physical structures of lignocellulosic biomass. However, compounds that inhibit both enzymes and microbial activity, including lignin-derived phenolics, soluble sugars, furan aldehydes, and weak acids, are also generated during pretreatment. Insoluble lignin, which predominantly remains within the pretreated solids, also acts as a significant inhibitor of cellulases during hydrolysis of cellulose. Exposed lignin, which is modified to be more recalcitrant to enzymes during pretreatment, adsorbs cellulase nonproductively and reduces the availability of active cellulase for hydrolysis of cellulose. Similarly, lignin-derived phenolics inhibit or deactivate cellulase and ß-glucosidase via irreversible binding or precipitation. Meanwhile, the performance of fermenting microorganisms is negatively affected by phenolics, sugar degradation products, and weak acids. This review describes the current knowledge regarding the contributions of inhibitors present in whole pretreatment slurries to the enzymatic hydrolysis of cellulose and fermentation. Furthermore, we discuss various biological strategies to mitigate the effects of these inhibitors on enzymatic and microbial activity to improve the lignocellulose-to-biofuel process robustness. While the inhibitory effect of lignin on enzymes can be relieved through the use of lignin blockers and by genetically engineering the structure of lignin or of cellulase itself, soluble inhibitors, including phenolics, furan aldehydes, and weak acids, can be detoxified by microorganisms or laccase.

Butyric acid production from red algae by a newly isolated Clostridium sp. S1.

OBJECTIVE: To produce butyric acid from red algae such as Gelidium amansii in which galactose is a main carbohydrate, microorganisms utilizing galactose and tolerating inhibitors in hydrolysis including levulinic acid and 5-hydroxymethylfurfural (HMF) are required. RESULTS: A newly isolated bacterium, Clostridium sp. S1 produced butyric acid not only from galactose as the sole carbon source but also from a mixture of galactose and glucose through simultaneous utilization. Notably, Clostridium sp. S1 produced butyric acid and a small amount of acetic acid with the butyrate:acetate ratio of 45.4:1 and it even converted acetate to butyric acid. Clostridium sp. S1 tolerated 0.5-2 g levulinic acid/l and recovered from HMF inhibition at 0.6-2.5 g/l, resulting in 85-92% butyric acid concentration of the control culture. When acid-pretreated G. amansii hydrolysate was used, Clostridium sp. S1 produced 4.83 g butyric acid/l from 10 g galactose/l and 1 g glucose/l. CONCLUSION: Clostridium sp. S1 produces butyric acid from red algae due to its characteristics in sugar utilization and tolerance to inhibitors, demonstrating its advantage as a red algae-utilizing microorganism.

Synthetic biology platform of CoryneBrick vectors for gene expression in Corynebacterium glutamicum and its application to xylose utilization.

Currently, the majority of tools in synthetic biology have been designed and constructed for model organisms such as Escherichia coli and Saccharomyces cerevisiae. In order to broaden the spectrum of organisms accessible to such tools, we established a synthetic biological platform, called CoryneBrick, for gene expression in Corynebacterium glutamicum as a set of E. coli-C. glutamicum shuttle vectors whose elements are interchangeable with BglBrick standard parts. C. glutamicum is an established industrial microorganism for the production of amino acids, proteins, and commercially promising chemicals. Using the CoryneBrick vectors, we showed various time-dependent expression profiles of a red fluorescent protein. This CoryneBrick platform was also applicable for two-plasmid expression systems with a conventional C. glutamicum expression vector. In order to demonstrate the practical application of the CoryneBrick vectors, we successfully reconstructed the xylose utilization pathway in the xylose-negative C. glutamicum wild type by fast BglBrick cloning methods using multiple genes encoding for xylose isomerase and xylulose kinase, resulting in a growth rate of 0.11 ± 0.004 h(-1) and a xylose uptake rate of 3.35 mmol/gDW/h when 1 % xylose was used as sole carbon source. Thus, CoryneBrick vectors were shown to be useful engineering tools in order to exploit Corynebacterium as a synthetic platform for the production of chemicals by controllable expression of the genes of interest.

Characterization of modular bifunctional processive endoglucanase Cel5 from Hahella chejuensis KCTC 2396.

Cel5 from marine Hahella chejuensis is composed of glycoside hydrolase family-5 (GH5) catalytic domain (CD) and two carbohydrate binding modules (CBM6-2). The enzyme was expressed in Escherichia coli and purified to homogeneity. The optimum endoglucanase and xylanase activities of recombinant Cel5 were observed at 65 °C, pH 6.5 and 55 °C, pH 5.5, respectively. It exhibited K m of 1.8 and 7.1 mg/ml for carboxymethyl cellulose and birchwood xylan, respectively. The addition of Ca(2+) greatly improved thermostability and endoglucanase activity of Cel5. The Cel5 retained 90 % of its endoglucanase activity after 24 h incubation in presence of 5 M concentration of NaCl. Recombinant Cel5 showed production of cellobiose after hydrolysis of cellulosic substrates (soluble/insoluble) and methylglucuronic acid substituted xylooligosaccharides after hydrolysis of glucuronoxylans by endo-wise cleavage. These results indicated that Cel5 as bifunctional enzyme having both processive endoglucanase and xylanase activities. The multidomain structure of Cel5 is clearly distinguished from the GH5 bifunctional glycoside hydrolases characterized to date, which are single domain enzymes. Sequence analysis and homology modeling suggested presence of two conserved binding sites with different substrate specificities in CBM6-2 and a single catalytic site in CD. Residues Glu132 and Glu219 were identified as key catalytic amino acids by sequence alignment and further verified by using site directed mutagenesis. CBM6-2 plays vital role in catalytic activity and thermostability of Cel5. The bifunctional activities and multiple substrate specificities of Cel5 can be utilized for efficient hydrolysis of cellulose and hemicellulose into soluble sugars.

Biosynthesis of pinene from glucose using metabolically-engineered Corynebacterium glutamicum.

Pinene is a monoterpenes (C10) that is produced in a genetically-engineered microbial host for its industrial applications in fragrances, flavoring agents, pharmaceuticals, and biofuels. Herein, we have metabolically-engineered Corynebacterium glutamicum, to produce pinene and studied its toxicity in C. glutamicum. Geranyl diphosphate synthases (GPPS) and pinene synthases (PS), obtained from Pinus taeda and Abies grandis, were co-expressed with over-expressed native 1-deoxy-d-xylulose-5-phosphate synthase (Dxs) and isopentenyl diphosphate isomerase (Idi) from C. glutamicum using CoryneBrick vector. Most strains expressing PS-GPPSs produced detectable amounts of pinene, but co-expression of DXS and IDI with PS (P. taeda) and GPPS (A. grandis) resulted in 27 µg ± 7 α-pinene g(-1) cell dry weight, which is the first report in C. glutamicum. Further engineering of PS and GPPS in the C. glutamicum strain may increase pinene production.

The regulation of 2,3-butanediol synthesis in Klebsiella pneumoniae as revealed by gene over-expressions and metabolic flux analysis.

A variety of microorganism species are able naturally to produce 2,3-butanediol (2,3-BDO), although only a few of them are suitable for consideration as having potential for mass production purposes. Klebsiella pneumoniae (K. pneumoniae) is one such strain which has been widely studied and used industrially to produce 2,3-BDO. In the central carbon metabolism of K. pneumoniae, the 2,3-BDO synthesis pathway is dominated by three essential enzymes, namely acetolactate decarboxylase, acetolactate synthase, and butanediol dehydrogenase, which are encoded by the budA, budB, and budC genes, respectively. The mechanisms of the three enzymes have been characterized with regard to their function and roles in 2,3-BDO synthesis and cell growth (Blomqvist et al. in J Bacteriol 175(5):1392-1404, 1993), while a few studies have focused on the cooperative mechanisms of the three enzymes and their mutual interactions. Therefore, the K. pneumoniae KCTC2242::ΔwabG wild-type strain was utilized to reconstruct seven new mutants by single, double, and triple overexpression of the three enzymes key to this study. Subsequently, continuous cultures were performed to obtain steady-state metabolism in the organisms and experimental data were analyzed by metabolic flux analysis (MFA) to determine the regulation mechanisms. The MFA results showed that the seven overexpressed mutants all exhibited enhanced 2,3-BDO production, and the strain overexpressing the budBA gene produced the highest yield. While the enzyme encoded by the budA gene produced branched-chain amino acids which were favorable for cell growth, the budB gene enzyme rapidly enhanced the conversion of acetolactate to acetoin in an oxygen-dependent manner, and the budC gene enzyme catalyzed the reversible conversion of acetoin to 2,3-BDO and regulated the intracellular NAD(+)/NADH balance.

Tailored polyhydroxyalkanoate production from renewable non-fatty acid carbon sources using engineered Cupriavidus necator H16.

As thermoplastic, nontoxic, and biocompatible polyesters, polyhydroxyalkanoates (PHAs) are considered promising biodegradable plastic candidates for diverse applications. Short-chain-length/medium-chain-length (SCL/MCL) PHA copolymers are flexible and versatile PHAs that are typically produced from fatty acids, which are expensive and toxic. Therefore, to achieve the sustainable biosynthesis of SCL/MCL-PHAs from renewable non-fatty acid carbon sources (e.g., sugar or CO2), we used the lithoautotrophic bacterium Cupriavidus necator H16 as a microbial platform. Specifically, we synthesized tailored PHA copolymers with varying MCL-3-hydroxyalkanoate (3HA) compositions (10-70 mol%) from fructose by rewiring the MCL-3HA biosynthetic pathways, including (i) the thioesterase-mediated free fatty acid biosynthetic pathway coupled with the beta-oxidation cycle and (ii) the hydroxyacyl transferase-mediated fatty acid de novo biosynthetic pathway. In addition to sugar-based feedstocks, engineered strains are also promising platforms for the lithoautotrophic production of SCL/MCL-PHAs from CO2. The set of engineered C. necator strains developed in this study provides greater opportunities to produce customized polymers with controllable monomer compositions from renewable resources.