Metabolic engineering of the acid-tolerant yeast Pichia kudriavzevii for efficient L-malic acid production at low pH.

Currently, the biological production of L-malic acid (L-MA) is mainly based on the fermentation of filamentous fungi at near-neutral pH, but this process requires large amounts of neutralizing agents, resulting in the generation of waste salts when free acid is obtained in the downstream process, and the environmental hazards associated with the waste salts limit the practical application of this process. To produce L-MA in a more environmentally friendly way, we metabolically engineered the acid-tolerant yeast Pichia kudriavzevii and achieved efficient production of L-MA through low pH fermentation. First, an initial L-MA-producing strain that relies on the reductive tricarboxylic acid (rTCA) pathway was constructed. Subsequently, the L-MA titer and yield were further increased by fine-tuning the flux between the pyruvate and oxaloacetate nodes. In addition, we found that the insufficient supply of NADH for cytoplasmic malate dehydrogenase (MDH) hindered the L-MA production at low pH, which was resolved by overexpressing the soluble pyridine nucleotide transhydrogenase SthA from E. coli. Transcriptomic and metabolomic data showed that overexpression of EcSthA contributed to the activation of the pentose phosphate pathway and provided additional reducing power for MDH by converting NADPH to NADH. Furthermore, overexpression of EcSthA was found to help reduce the accumulation of the by-product pyruvate but had no effect on the accumulation of succinate. In microaerobic batch fermentation in a 5-L fermenter, the best strain, MA009-10-URA3 produced 199.4 g/L L-MA with a yield of 0.94 g/g glucose (1.27 mol/mol), with a productivity of 1.86 g/L/h. The final pH of the fermentation broth was approximately 3.10, meaning that the amount of neutralizer used was reduced by more than 50% compared to the common fermentation processes using filamentous fungi. To our knowledge, this is the first report of the efficient bioproduction of L-MA at low pH and represents the highest yield of L-MA in yeasts reported to date.

Metabolic engineering of Saccharomyces cerevisiae for gram-scale diosgenin production.

Diosgenin (DSG) is a naturally occurring steroidal saponin with a variety of biological activities that is also an important precursor for the synthesis of various steroidal drugs. The traditional industrial production of DSG is based on natural plant extraction and chemical processing. However, the whole process is time-consuming, laborious, and accompanied by severe environmental pollution. Therefore, it is necessary to develop a more convenient and environmentally-friendly process to realize the green production of DSG. In our previous work, we achieved de novo synthesis of DSG in Saccharomyces cerevisiae using glucose as the carbon source. However, DSG production was only at the milligram level, which is too low for industrial production. In this work, we further developed yeast strains for DSG overproduction by optimizing the synthesis pathway, fine-tuning pathway gene expression, and eliminating competing pathways. Cholesterol 22-hydroxylase was used to construct the DSG biosynthesis pathway. The optimal ratio of cytochrome P450 (CYP) to cytochrome P450 reductase (CPR) associated with DSG synthesis was screened to increase DSG production. Weakening the expression of the ERG6 gene further increased DSG synthesis and reduced the formation of by-products. In addition, we investigated the impact of DSG accumulation on yeast cell physiology and growth by transcriptome analysis and found that the multidrug transporter PDR5 and the sterol-binding protein PRY1 contributed to DSG production. Finally, we obtained a DSG titer of 2.03 g/L after 288 h of high-cell-density fed-batch fermentation using the engineered strain LP118, which represents the highest DSG titer reported to date for a yeast de novo synthesis system.

A CRISPR-based chromosomal-separation technique for Escherichia coli.

BACKGROUND: Natural life systems can be significantly modified at the genomic scale by human intervention, demonstrating the great innovation capacity of genome engineering. Large epi-chromosomal DNA structures were established in Escherichia coli cells, but some of these methods were inconvenient, using heterologous systems, or relied on engineered E. coli strains. RESULTS: The wild-type model bacterium E. coli has a single circular chromosome. In this work, a novel method was developed to split the original chromosome of wild-type E. coli. With this method, novel E. coli strains containing two chromosomes of 0.10 Mb and 4.54 Mb, and 2.28 Mb and 2.36 Mb were created respectively, designated as E. coli0.10/4.54 and E. coli2.28/2.36. The new chromosomal arrangement was proved by PCR amplification of joint regions as well as a combination of Nanopore and Illumina sequencing analysis. While E. coli0.10/4.54 was quite stable, the two chromosomes of E. coli2.28/2.36 population recombined into a new chromosome (Chr.4.64MMut), via recombination. Both engineered strains grew slightly slower than the wild-type, and their cell shapes were obviously elongated. CONCLUSION: Finally, we successfully developed a simple CRISPR-based genome engineering technique for the construction of multi-chromosomal E. coli strains with no heterologous genetic parts. This technique might be applied to other prokaryotes for synthetic biology studies and applications in the future.

Microbial adaptive evolution.

Bacterial species can adapt to significant changes in their environment by mutation followed by selection, a phenomenon known as "adaptive evolution." With the development of bioinformatics and genetic engineering, research on adaptive evolution has progressed rapidly, as have applications of the process. In this review, we summarize various mechanisms of bacterial adaptive evolution, the technologies used for studying it, and successful applications of the method in research and industry. We particularly highlight the contributions of Dr. L. O. Ingram. Microbial adaptive evolution has significant impact on our society not only from its industrial applications, but also in the evolution, emergence, and control of various pathogens.

Fine-tuning the expression of pathway gene in yeast using a regulatory library formed by fusing a synthetic minimal promoter with different Kozak variants.

BACKGROUND: Tailoring gene expression to balance metabolic fluxes is critical for the overproduction of metabolites in yeast hosts, and its implementation requires coordinated regulation at both transcriptional and translational levels. Although synthetic minimal yeast promoters have shown many advantages compared to natural promoters, their transcriptional strength is still limited, which restricts their applications in pathway engineering. RESULTS: In this work, we sought to expand the application scope of synthetic minimal yeast promoters by enhancing the corresponding translation levels using specific Kozak sequence variants. Firstly, we chose the reported UASF-E-C-Core1 minimal promoter as a library template and determined its Kozak motif (K0). Next, we randomly mutated the K0 to generate a chimeric promoter library, which was able to drive green fluorescent protein (GFP) expression with translational strengths spanning a 500-fold range. A total of 14 chimeric promoters showed at least two-fold differences in GFP expression strength compared to the K0 control. The best one named K528 even showed 8.5- and 3.3-fold increases in fluorescence intensity compared with UASF-E-C-Core1 and the strong native constitutive promoter PTDH3, respectively. Subsequently, we chose three representative strong chimeric promoters (K540, K536, and K528) from this library to regulate pathway gene expression. In conjunction with the tHMG1 gene for squalene production, the K528 variant produced the best squalene titer of 32.1 mg/L in shake flasks, which represents a more than 10-fold increase compared to the parental K0 control (3.1 mg/L). CONCLUSIONS: All these results demonstrate that this chimeric promoter library developed in this study is an effective tool for pathway engineering in yeast.

Directed evolution of alditol oxidase for the production of optically pure D-glycerate from glycerol in the engineered Escherichia coli.

D-glycerate is an attractive chemical for a wide variety of pharmaceutical, cosmetic, biodegradable polymers, and other applications. Now several studies have been reported about the synthesis of glycerate by different biotechnological and chemical routes from glycerol or other feedstock. Here, we present the construction of an Escherichia coli engineered strain to produce optically pure D-glycerate by oxidizing glycerol with an evolved variant of alditol oxidase (AldO) from Streptomyces coelicolor. This is achieved by starting from a previously reported variant mAldO and employing three rounds of directed evolution, as well as the combination of growth-coupled high throughput selection with colorimetric screening. The variant eAldO3-24 displays a higher substrate affinity toward glycerol with 5.23-fold than the wild-type AldO, and a 1.85-fold increase of catalytic efficiency (kcat/KM). Then we introduced an isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible T7 expression system in E. coli to overexpress the variant eAldO3-24, and deleted glucosylglycerate phosphorylase encoding gene ycjM to block the consumption of D-glycerate. Finally, the resulting strain TZ-170 produced 30.1 g/l D-glycerate at 70 h with a yield of 0.376 mol/mol in 5-l fed-batch fermentation.

Identification of Absidia orchidis steroid 11ß-hydroxylation system and its application in engineering Saccharomyces cerevisiae for one-step biotransformation to produce hydrocortisone.

Hydrocortisone is an effective anti-inflammatory drug and also an important intermediate for synthesis of other steroid drugs. The filamentous fungus Absidia orchidis is renowned for biotransformation of acetylated cortexolone through 11ß-hydroxylation to produce hydrocortisone. However, due to the presence of 11α-hydroxylase in A. orchidis, the 11α-OH by-product epi-hydrocortisone is always produced in a 1:1 M ratio with hydrocortisone. In order to decrease epi-hydrocortisone production, Saccharomyces cerevisiae was engineered in this work as an alternative way to produce hydrocortisone through biotransformation. Through transcriptomic analysis coupled with genetic verification in S. cerevisiae, the A. orchidis steroid 11ß-hydroxylation system was characterized, including a cytochrome P450 enzyme CYP5311B2 and its associated redox partners cytochrome P450 reductase and cytochrome b5. CYP5311B2 produces a mix of stereoisomers containing 11ß- and 11α-hydroxylation derivatives in a 4:1 M ratio. This fungal steroid 11ß-hydroxylation system was reconstituted in S. cerevisiae for hydrocortisone production, resulting in a productivity of 22 mg/L·d. Protein engineering of CYP5311B2 generated a R126D/Y398F variant, which had 3 times higher hydrocortisone productivity compared to the wild type. Elimination of C20-hydroxylation by-products and optimization of the expression of A. orchidis 11ß-hydroxylation system genes further increased hydrocortisone productivity by 238% to 223 mg/L·d. In addition, a novel steroid transporter ClCDR4 gene was identified from Cochliobolus lunatus, overexpression of which further increased hydrocortisone productivity to 268 mg/L·d in S. cerevisiae. Through increasing cell mass, 1060 mg/L hydrocortisone was obtained in 48 h and the highest productivity reached 667 mg/L·d. This is the highest hydrocortisone titer reported for yeast biotransformation system so far.

Construction of a carbon-conserving pathway for glycolate production by synergetic utilization of acetate and glucose in Escherichia coli.

Glycolate is a bulk chemical which has been widely used in textile, food processing, and pharmaceutical industries. Glycolate can be produced from sugars by microbial fermentation. However, when using glucose as the sole carbon source, the theoretical maximum carbon molar yield of glycolate is 0.67 mol/mol due to the loss of carbon as CO2. In this study, a synergetic system for simultaneous utilization of acetate and glucose was designed to increase the carbon yield. The main function of glucose is to provide NADPH while acetate to provide the main carbon backbone for glycolate production. Theoretically, 1 glucose and 5 acetate can produce 6 glycolate, and the carbon molar yield can be increased to 0.75 mol/mol. The whole synthetic pathway was divided into two modules, one for converting acetate to glycolate and another to utilize glucose to provide NADPH. After engineering module I through activation of acs, gltA, aceA and ycdW, glycolate titer increased from 0.07 to 2.16 g/L while glycolate yields increased from 0.04 to 0.35 mol/mol-acetate and from 0.03 to 1.04 mol/mol-glucose. Module II was then engineered to increase NADPH supply. Through deletion of pfkA, pfkB, ptsI and sthA genes as well as upregulating zwf, pgl and tktA, glycolate titer increased from 2.16 to 4.86 g/L while glycolate yields increased from 0.35 to 0.82 mol/mol-acetate and from 1.04 to 6.03 mol/mol-glucose. The activities of AceA and YcdW were further increased to pull the carbon flux to glycolate, which increased glycolate yield from 0.82 to 0.92 mol/mol-acetate. Fed-batch fermentation of the final strain NZ-Gly303 produced 73.3 g/L glycolate with a productivity of 1.04 g/(L·h). The acetate to glycolate yield was 0.85 mol/mol (1.08 g/g), while glucose to glycolate yield was 6.1 mol/mol (2.58 g/g). The total carbon molar yield was 0.60 mol/mol, which reached 80% of the theoretical value.

Elucidation of the complete biosynthetic pathway of the main triterpene glycosylation products of Panax notoginseng using a synthetic biology platform.

UDP-glycosyltransferase (UGT)-mediated glycosylation is a widespread modification of plant natural products (PNPs), which exhibit a wide range of bioactivities, and are of great pharmaceutical, ecological and agricultural significance. However, functional annotation is available for less than 2% of the family 1 UGTs, which currently has 20,000 members that are known to glycosylate several classes of PNPs. This low percentage illustrates the difficulty of experimental study and accurate prediction of their function. Here, a synthetic biology platform for elucidating the UGT-mediated glycosylation process of PNPs was established, including glycosyltransferases dependent on UDP-glucose and UDP-xylose. This platform is based on reconstructing the specific PNPs biosynthetic pathways in dedicated microbial yeast chassis by the simple method of plug-and-play. Five UGT enzymes were identified as responsible for the biosynthesis of the main glycosylation products of triterpenes in Panax notoginseng, including a novel UDP-xylose dependent glycosyltransferase enzyme for notoginsenoside R1 biosynthesis. Additionally, we constructed a yeast cell factory that yields >1 g/L of ginsenoside compound K. This platform for functional gene identification and strain engineering can serve as the basis for creating alternative sources of important natural products and thereby protecting natural plant resources.

Identification of a novel cytochrome P450 enzyme that catalyzes the C-2α hydroxylation of pentacyclic triterpenoids and its application in yeast cell factories.

C-2α hydroxylated triterpenoids are a large class of plant secondary metabolites. These compounds, such as maslinic, corosolic and alphitolic acid, have important biological activities against HIV, cancer and diabetes. However, the biosynthesis pathways of these compounds have not been completely elucidated. Specifically, the cytochrome P450 (CYP) enzyme responsible for C-2α hydroxylation was unknown. In this study, a novel CYP enzyme that catalyzes C-2α hydroxylation was identified in Crataegus pinnatifida (Hawthorn) using a metabolic engineering platform. It is a multifunctional enzyme with C-2α oxidase activity on oleanane-, ursane- and lupane-type pentacyclic triterpenoids. In addition, the complete biosynthesis pathways of these three triterpenoids were reconstituted in yeast, resulting in the production of 384, 141 and 23 mg/L of maslinic, corosolic and alphitolic acid, respectively. This metabolic engineering platform for functional gene identification and strain engineering can serve as the basis for creating alternative pathways for the microbial production of important natural products.

Production of 14α-hydroxysteroids by a recombinant Saccharomyces cerevisiae biocatalyst expressing of a fungal steroid 14α-hydroxylation system.

The 14α-hydroxysteroids have specific anti-gonadotropic and carcinolytic biological activities and can be produced by microbial biotransformation. The steroid 11ß-/14α-hydroxylase P-450lun from Cochliobolus lunatus is the only fungal cytochrome P450 enzyme identified to date with steroid C14 hydroxylation ability. Previous work has mainly revealed the 11ß-hydroxylation activity of the P-450lun towards cortexolone (RSS) substrate; however, the potential steroid 14α-hydroxylation activity of this enzyme, especially for androstenedione (AD) substrate, has not yet conducted in-depth testing. In this work, we further tested the steroid 14α-hydroxylation activity of the P-450lun towards RSS and AD in the Saccharomyces cerevisiae system. We demonstrated that P-450lun functions as the specific 14α-hydroxylase towards the AD substrate (regiospecificity > 99%); however, it showed a poor C14-hydroxylation regiospecificity (around 40%) for the RSS substrate. In addition, through transcriptome analysis combined with gene functional characterizations, we also identified and cloned the gene for the P-450lun-associated redox partner CPRlun. Finally, through codon optimization, knockout of genes for the side reactions related enzymes GCY1 and YPR1, and increasing copies of the P-450lun and CPRlun, we developed a recombinant S. cerevisiae biocatalyst based on the C. lunatus steroid 14α-hydroxylation system to produce 14α-hydroxysteroids. Initial production of 14α-OH-AD (150 mg/L day productivity, 99% regioisomeric purity, and 60% w/w yield) and 14α-OH-RSS (64 mg/L day productivity, 40% regioisomeric purity, and 26% w/w yield) were separately achieved in shake flasks; these results represent the highest level of 14α-hydroxysteroid production in the current yeast system.

A novel point mutation in RpoB improves osmotolerance and succinic acid production in Escherichia coli.

BACKGROUND: Escherichia coli suffer from osmotic stress during succinic acid (SA) production, which reduces the performance of this microbial factory. RESULTS: Here, we report that a point mutation leading to a single amino acid change (D654Y) within the ß-subunit of DNA-dependent RNA polymerase (RpoB) significantly improved the osmotolerance of E. coli. Importation of the D654Y mutation of RpoB into the parental strain, Suc-T110, increased cell growth and SA production by more than 40% compared to that of the control under high glucose osmolality. The transcriptome profile, determined by RNA-sequencing, showed two distinct stress responses elicited by the mutated RpoB that counterbalanced the osmotic stress. Under non-stressed conditions, genes involved in the synthesis and transport of compatible solutes such as glycine-betaine, glutamate or proline were upregulated even without osmotic stimulation, suggesting a "pre-defense" mechanism maybe formed in the rpoB mutant. Under osmotic stressed conditions, genes encoding diverse sugar transporters, which should be down-regulated in the presence of high osmotic pressure, were derepressed in the rpoB mutant. Additional genetic experiments showed that enhancing the expression of the mal regulon, especially for genes that encode the glycoporin LamB and maltose transporter, contributed to the osmotolerance phenotype. CONCLUSIONS: The D654Y single amino acid substitution in RpoB rendered E. coli cells resistant to osmotic stress, probably due to improved cell growth and viability via enhanced sugar uptake under stressed conditions, and activated a potential "pre-defense" mechanism under non-stressed conditions. The findings of this work will be useful for bacterial host improvement to enhance its resistance to osmotic stress and facilitate bio-based organic acids production.

The CRISPR/Cas9-facilitated multiplex pathway optimization (CFPO) technique and its application to improve the Escherichia coli xylose utilization pathway.

One of the most important research subjects of metabolic engineering is the pursuit of balanced metabolic pathways, which requires the modulation of expression of many genes. However, simultaneously modulating multiple genes on the chromosome remains challenging in prokaryotic organisms, including the industrial workhorse - Escherichia coli. In this work, the CRISPR/Cas9-facilitated multiplex pathway optimization (CFPO) technique was developed to simultaneously modulate the expression of multiple genes on the chromosome. To implement it, two plasmids were employed to target Cas9 to regulatory sequences of pathway genes, and a donor DNA plasmid library was constructed containing a regulator pool to modulate the expression of these genes. A modularized plasmid construction strategy was used to enable the assembly of a complex donor DNA plasmid library. After genome editing using this technique, a combinatorial library was obtained with variably expressed pathway genes. As a demonstration, the CFPO technique was applied to the xylose metabolic pathway genes in E. coli to improve xylose utilization. Three transcriptional units containing a total of four genes were modulated simultaneously with 70% efficiency, and improved strains were selected from the resulting combinatorial library by growth enrichment. The best strain, HQ304, displayed a 3-fold increase of the xylose-utilization rate. Finally, the xylose-utilization pathway of HQ304 was analyzed enzymologically to determine the optimal combination of enzyme activities.

Balanced activation of IspG and IspH to eliminate MEP intermediate accumulation and improve isoprenoids production in Escherichia coli.

The MEP pathway genes were modulated to investigate whether there were new rate-limiting steps and toxic intermediates in this pathway. Activating IspG led to significant decrease of cell growth and ß-carotene production. It was found that ispG overexpression led to accumulation of intermediate HMBPP, which seriously interfered with synthesis machinery of nucleotide and protein in Escherichia coli. Activation of the downstream enzyme IspH could solve HMBPP accumulation problem and eliminate the negative effects of ispG overexpression. In addition, intermediate MECPP accumulated in the starting strain, while balanced activation of IspG and IspH could push the carbon flux away from MECPP and led to 73% and 77% increase of ß-carotene and lycopene titer respectively. Our work for the first time identified HMBPP to be a cytotoxic intermediate in MEP pathway and demonstrated that balanced activation of IspG and IspH could eliminate accumulation of HMBPP and MECPP and improve isoprenoids production.

Osmotolerance in Escherichia coli Is Improved by Activation of Copper Efflux Genes or Supplementation with Sulfur-Containing Amino Acids.

Improvement in the osmotolerance of Escherichia coli is essential for the production of high titers of various bioproducts. In this work, a cusS mutation that was identified in the previously constructed high-succinate-producing E. coli strain HX024 was investigated for its effect on osmotolerance. CusS is part of the two-component system CusSR that protects cells from Ag(I) and Cu(I) toxicity. Changing cusS from strain HX024 back to its original sequence led to a 24% decrease in cell mass and succinate titer under osmotic stress (12% glucose). When cultivated with a high initial glucose concentration (12%), introduction of the cusS mutation into parental strain Suc-T110 led to a 21% increase in cell mass and a 40% increase in succinate titer. When the medium was supplemented with 30 g/liter disodium succinate, the cusS mutation led to a 120% increase in cell mass and a 492% increase in succinate titer. Introducing the cusS mutation into the wild-type strain ATCC 8739 led to increases in cell mass of 87% with 20% glucose and 36% using 30 g/liter disodium succinate. The cusS mutation increased the expression of cusCFBA, and gene expression levels were found to be positively related to osmotolerance abilities. Because high osmotic stress has been associated with deleterious accumulation of Cu(I) in the periplasm, activation of CusCFBA may alleviate this effect by transporting Cu(I) out of the cells. This hypothesis was confirmed by supplementing sulfur-containing amino acids that can chelate Cu(I). Adding methionine or cysteine to the medium increased the osmotolerance of E. coli under anaerobic conditions.IMPORTANCE In this work, an activating Cus copper efflux system was found to increase the osmotolerance of E. coli In addition, new osmoprotectants were identified. Supplementation with methionine or cysteine led to an increase in osmotolerance of E. coli under anaerobic conditions. These new strategies for improving osmotolerance will be useful for improving the production of chemicals in industrial bioprocesses.

The transcription cofactor FgSwi6 plays a role in growth and development, carbendazim sensitivity, cellulose utilization, lithium tolerance, deoxynivalenol production and virulence in the filamentous fungus Fusarium graminearum.

Fusarium head blight (FHB) is a worldwide devastating disease of wheat, barley and other small grain cereals and caused primarily by Fusarium graminearum. Carbendazim (MBC) is one of the fungicides widely used to control FHB in China. In the present study, we have identified the F. graminearum locus FGSG_04220 as the sequence homolog for Saccharomyces cerevisiae ScSWI6, named FgSWI6 hereafter. Deletion of FgSWI6 causes mycelium of F. graminearum become sensitive to MBC in liquid medium. In addition, deletion of FgSWI6 reduces mycelial growth as well as production and development of conidia. F. graminearum cells lacking FgSWI6 show reduced production efficiency and sizes of perithecia as well as a defect in the production of ascus and ascospore. FgSWI6 is required for the cellulose utilization, lithium tolerance and deoxynivalenol (DON) production of this pathogen. Furthermore, deletion of FgSWI6 significantly attenuates the virulence of F. graminearum on wheat. Therefore, FgSwi6p plays an important role in growth and development of the economically important fungal pathogen F. graminearum as well as its resistance to MBC.

[Sterol transport proteins in yeast: a review].

Sterols are a class of cyclopentano-perhydrophenanthrene derivatives widely present in living organisms. Sterols are important components of cell membranes. In addition, they also have important physiological and pharmacological activities. With the development of synthetic biology and metabolic engineering technology, yeast cells are increasingly used for the heterologous synthesis of sterols in recent years. Nevertheless, since sterols are hydrophobic macromolecules, they tend to accumulate in the membrane fraction of yeast cells and consequently trigger cytotoxicity, which hampers the further improvement of sterols yield. Therefore, revealing the mechanism of sterol transport in yeast, especially understanding the working principle of sterol transporters, is vital for designing strategies to relieve the toxicity of sterol accumulation and increasing sterol yield in yeast cell factories. In yeast, sterols are mainly transported through protein-mediated non-vesicular transport mechanisms. This review summarizes five types of sterol transport-related proteins that have been reported in yeast, namely OSBP/ORPs family proteins, LAM family proteins, ABC transport family proteins, CAP superfamily proteins, and NPC-like sterol transport proteins. These transporters play important roles in intracellular sterol gradient distribution and homeostasis maintenance. In addition, we also review the current status of practical applications of sterol transport proteins in yeast cell factories.

Artificial Diploid Escherichia coli by a CRISPR Chromosome-Doubling Technique.

Synthetic biology has been represented by the creation of artificial life forms at the genomic scale. In this work, a CRISPR-based chromosome-doubling technique is designed to first construct an artificial diploid Escherichia coli cell. The stable single-cell diploid E. coli is isolated by both maximal dilution plating and flow cytometry, and confirmed with quantitative PCR, fluorescent in situ hybridization, and third-generation genome sequencing. The diploid E. coli has a greatly reduced growth rate and elongated cells at 4-5 µm. It is robust against radiation, and the survival rate after exposure to UV increased 40-fold relative to WT. As a novel life form, the artificial diploid E. coli is an ideal substrate for research fundamental questions in life science concerning polyploidy. And this technique may be applied to other bacteria.

Development of CRISPR/Cas9-Based Genome Editing Tools for Polyploid Yeast Cyberlindnera jadinii and Its Application in Engineering Heterologous Steroid-Producing Strains.

In this study, a suite of efficient CRISPR/Cas9 tools was developed to overcome the genetic manipulation challenges posed by the polyploid genome of industrial yeast Cyberlindnera jadinii. The developed CRISPR/Cas9 system can achieve a 100% single-gene knockdown efficiency in strain NBRC0988. Moreover, the integration of a single exogenous gene into the target locus using a 50 bp homology arm achieved near-100% efficiency. The efficiency of simultaneous integration of three genes into the chromosome is strongly influenced by the length of the homology arm, with the highest integration efficiency of 62.5% obtained when selecting a homology arm of about 500 bp. By utilizing the CRISPR/Cas system, this study demonstrated the potential of C. jadinii in producing heterologous sterols. Through shake-flask fermentation, the engineered strains produced 92.1 and 81.8 mg/L of campesterol and cholesterol, respectively. Furthermore, the production levels of these two sterols were further enhanced through high-cell-density fed-batch fermentation in a 5 L bioreactor. The highest titer of campesterol reached 807 mg/L [biomass OD600 = 294, productivity of 6.73 mg/(L·h)]. The titer of cholesterol reached 1.52 g/L [biomass OD600 = 380, productivity of 9.06 mg/(L·h)], marking the first gram-scale production of steroidal compounds in C. jadinii.

[Green biosynthesis of chiral pharmaceutical chemicals].

In nature, chirality is a common phenomenon and closely related to life, also significantly influences the properties of the substance. The chemical synthesis of chiral pharmaceutical chemicals has encountered challenges such as poor atom economy and process economy, serious environmental pollution and waste of the resource. The biosynthesis route has the advantages of high selectivity and environmental-friendliness. In recent years, the rapid developments in the accessible key enzymes, understanding of catalytic mechanism, construction of new synthetic pathways of optical pure intermediates, process development and scale-up production have made it possible to address the challenges encountered in the production of active pharmaceutical ingredients, thus promoting a green and sustainable pharmaceutical industry in China. This review summarized the achievements made in this field by researchers at Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences.