Fine-tuning Bacterial Cyclic di-AMP Production for Durable Antitumor Effects Through the Activation of the STING Pathway.

The stimulator of interferon genes (STING) protein is an important and promising innate immune target for tumor therapy. However, the instability of the agonists of STING and their tendency to cause systemic immune activation is a hurdle. The STING activator, cyclic di-adenosine monophosphate (CDA), produced by the modified Escherichia coli Nissle 1917, shows high antitumor activity and effectively reduces the systemic effects of the "off-target" caused by the activation of the STING pathway. In this study, we used synthetic biological approaches to optimize the translation levels of the diadenylate cyclase that catalyzes CDA synthesis in vitro. We developed 2 engineered strains, CIBT4523 and CIBT4712, for producing high levels of CDA while keeping their concentrations within a range that did not compromise the growth. Although CIBT4712 exhibited stronger induction of the STING pathway corresponding to in vitro CDA levels, it had lower antitumor activity than CIBT4523 in an allograft tumor model, which might be related to the stability of the surviving bacteria in the tumor tissue. CIBT4523 exhibited complete tumor regression, prolonged survival of mice, and rejection of rechallenged tumors, thus, offering new possibilities for more effective tumor therapy. We showed that the appropriate production of CDA in engineered bacterial strains is essential for balancing antitumor efficacy and self-toxicity.

Expression of phenylalanine ammonia lyase as an intracellularly free and extracellularly cell surface-immobilized enzyme on a gut microbe as a live biotherapeutic for phenylketonuria.

Phenylketonuria (PKU), a disease resulting in the disability to degrade phenylalanine (Phe) is an inborn error with a 1 in 10,000 morbidity rate on average around the world which leads to neurotoxicity. As an potential alternative to a protein-restricted diet, oral intake of engineered probiotics degrading Phe inside the body is a promising treatment, currently at clinical stage II (Isabella, et al., 2018). However, limited transmembrane transport of Phe is a bottleneck to further improvement of the probiotic's activity. Here, we achieved simultaneous degradation of Phe both intracellularly and extracellularly by expressing genes encoding the Phe-metabolizing enzyme phenylalanine ammonia lyase (PAL) as an intracellularly free and a cell surface-immobilized enzyme in Escherichia coli Nissle 1917 (EcN) which overcomes the transportation problem. The metabolic engineering strategy was also combined with strengthening of Phe transportation, transportation of PAL-catalyzed trans-cinnamic acid and fixation of released ammonia. Administration of our final synthetic strain TYS8500 with PAL both displayed on the cell surface and expressed inside the cell to the PahF263S PKU mouse model reduced blood Phe concentration by 44.4% compared to the control EcN, independent of dietary protein intake. TYS8500 shows great potential in future applications for PKU therapy.

Genome Editing of Corynebacterium glutamicum Using CRISPR-Cpf1 System.

Corynebacterium glutamicum, as an important microbial chassis, has great potential in industrial application. However, complicated genetic modification is severely slowed by lack of efficient genome editing tools. The Streptococcus pyogenes (Sp) CRISPR-Cas9 system has been verified as a very powerful tool for mediating genome alteration in many microorganisms but cannot work well in C. glutamicum. We recently developed two Francisella novicida (Fn) CRISPR-Cpf1 assisted systems for genome editing via homologous recombination in C. glutamicum. Here, we describe the protocols and demonstrated that N iterative rounds of genome editing can be achieved in 3 N + 4 or 3 N + 2 days, respectively.

Reducing glucoamylase usage for commercial-scale ethanol production from starch using glucoamylase expressing Saccharomyces cerevisiae.

The development of yeast that converts raw corn or cassava starch to ethanol without adding the exogenous α-amylase and/or glucoamylase would reduce the overall ethanol production cost. In this study, two copies of codon-optimized Saccharomycopsis fibuligera glucoamylase genes were integrated into the genome of the industrial Saccharomyces cerevisiae strain CCTCC M94055, and the resulting strain CIBTS1522 showed comparable basic growth characters with the parental strain. We systemically evaluated the fermentation performance of the CIBTS1522 strain using the raw corn or cassava starch at small and commercial-scale, and observed that a reduction of at least 40% of the dose of glucoamylase was possible when using the CIBTS1522 yeast under real ethanol production condition. Next, we measured the effect of the nitrogen source, the phosphorous source, metal ions, and industrial microbial enzymes on the strain's cell wet weight and ethanol content, the nitrogen source and acid protease showed a positive effect on these parameters. Finally, orthogonal tests for some other factors including urea, acid protease, inoculum size, and glucoamylase addition were conducted to further optimize the ethanol production. Taken together, the CIBTS1522 strain was identified as an ideal candidate for the bioethanol industry and a better fermentation performance could be achieved by modifying the industrial culture media and condition.

De Novo Engineering of Corynebacterium glutamicum for l-Proline Production.

l-Proline is an important amino acid that has various industrial applications. Industrial l-proline-producing strains are obtained by the mutagenesis of Corynebacterium glutamicum. In this study, the optimized C. glutamicum genome-editing tools were further applied in the de novo construction of a hyper-l-proline-producing strain. Overexpression of a feedback inhibition-resistant γ-glutamic kinase mutant ProBG149K, deletion of a proline dehydrogenase to block l-proline degradation, overexpression of glutamate dehydrogenase to increase glutamate synthesis flux, the mutation of 6-phosphate gluconate dehydrogenase and glucose-6-phosphate-dehydrogenase in the pentose phosphate pathway to enhance NADPH supply, the deletion of pyruvate aminotransferase to decrease the byproduct l-alanine synthesis, and weakening of α-ketoglutarate dehydrogenase to regulate the TCA cycle were combined to obtain ZQJY-9. ZQJY-9 produced 19.68 ± 0.22 g/L of l-proline in flask fermentation and was also demonstrated at the 3 L bioreactor level by fed-batch fermentation producing 120.18 g/L of l-proline at 76 h with the highest productivity of 1.581 g/L/h.

CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum.

Corynebacterium glutamicum is an important industrial metabolite producer that is difficult to genetically engineer. Although the Streptococcus pyogenes (Sp) CRISPR-Cas9 system has been adapted for genome editing of multiple bacteria, it cannot be introduced into C. glutamicum. Here we report a Francisella novicida (Fn) CRISPR-Cpf1-based genome-editing method for C. glutamicum. CRISPR-Cpf1, combined with single-stranded DNA (ssDNA) recombineering, precisely introduces small changes into the bacterial genome at efficiencies of 86-100%. Large gene deletions and insertions are also obtained using an all-in-one plasmid consisting of FnCpf1, CRISPR RNA, and homologous arms. The two CRISPR-Cpf1-assisted systems enable N iterative rounds of genome editing in 3N+4 or 3N+2 days. A proof-of-concept, codon saturation mutagenesis at G149 of γ-glutamyl kinase relieves L-proline inhibition using Cpf1-assisted ssDNA recombineering. Thus, CRISPR-Cpf1-based genome editing provides a highly efficient tool for genetic engineering of Corynebacterium and other bacteria that cannot utilize the Sp CRISPR-Cas9 system.

Construction of fast xylose-fermenting yeast based on industrial ethanol-producing diploid Saccharomyces cerevisiae by rational design and adaptive evolution.

BACKGROUND: It remains a challenge for recombinant S. cerevisiae to convert xylose in lignocellulosic biomass hydrolysates to ethanol. Although industrial diploid strains are more robust compared to laboratory haploid strains, however, industrial diploid S. cerevisiae strains have been less pursued in previous studies. This work aims to construct fast xylose-fermenting yeast using an industrial ethanol-producing diploid S. cerevisiae strain as a host. RESULTS: Fast xylose-fermenting yeast was constructed by genome integration of xylose-utilizing genes and adaptive evolution, including 1) Piromyces XYLA was introduced to enable the host strain to convert xylose to xylulose; 2) endogenous genes (XKS1, RKI1, RPE1, TKL1, and TAL1) were overexpressed to accelerate conversion of xylulose to ethanol; 3) Candida intermedia GXF1, which encodes a xylose transporter, was introduced at the GRE3 locus to improve xylose uptake; 4) aerobic evolution in rich xylose media was carried out to increase growth and xylose consumption rates. The best evolved strain CIBTS0735 consumed 80 g/l glucose and 40 g/l xylose in rich media within 24 hours at an initial OD600 of 1.0 (0.63 g DCW/l) and produced 53 g/l ethanol. CONCLUSIONS: Based on the above fermentation performance, we conclude that CIBTS0735 shows great potential for ethanol production from lignocellulosic biomass.