Building a highly efficient Streptomyces super-chassis for secondary metabolite production by reprogramming naturally-evolved multifaceted shifts.

Streptomyces has an extensive array of bioactive secondary metabolites (SMs). Nevertheless, devising a framework for the heterologous production of these SMs remains challenging. We here reprogrammed a versatile plug-and-play Streptomyces super-chassis and established a universal pipeline for production of diverse SMs via understanding of the inherent pleiotropic effects of ethanol shock on jadomycin production in Streptomyces venezuelae. We initially identified and characterized a set of multiplex targets (afsQ1, bldD, bldA, and miaA) that contribute to SM (jadomycin) production when subjected to ethanol shock. Subsequently, we developed an ethanol-induced orthogonal amplification system (EOAS), enabling dynamic and precise control over targets. Ultimately, we integrated these multiplex targets into functional units governed by the EOAS, generating a universal and plug-and-play Streptomyces super-chassis. In addition to achieving the unprecedented titer and yield of jadomycin B, we also evidenced the potential of this super-chassis for production of diverse heterologous SMs, including antibiotic oxytetracycline, anticancer drug doxorubicins, agricultural herbicide thaxtomin A, and plant growth regulator guvermectin, all with the yields of >10 mg/g glucose in a simple mineral medium. Given that the production of SMs all required complexed medium and the cognate yields were usually much lower, our achievement of using a universal super-chassis and engineering pipeline in a simple mineral medium is promising for convenient heterologous production of SMs.

Extraction methods optimization of available heavy metals and the health risk assessment of the suburb soil in China.

Heavy metal pollution has attracted increasing concern due to its high toxicity and persistence. A suitable extraction procedure for available heavy metals in soil is necessary for assessing the ecological risk. In this work, the single extraction methods aided by shaking and microwaves were investigated and analyzed for their ability to extract available heavy metals from soil samples, and a total of 42 soil samples were collected from suburbs of Zhengzhou city in China. The extraction efficiency of Cu, Zn, As, and Cd in the certified fluvo-aquic soil was compared using eight different types of solutions: CaCl2, CH3COONH4, NH4NO3, CH3COOH, Na2EDTA, DTPA, HNO3, and NH4H2PO4. Results indicated that the shaking-assisted method that utilized Na2EDTA as an extractant demonstrated satisfactory efficiency and was chosen for further optimization and that the optimal conditions were obtained using 0.05 M Na2EDTA at pH 7, soil-liquid ratio 1:20, and extraction duration 2 h, which gained the perfect extraction efficiency ranging from 85.8 to 109.5%. The proposed approach has been applied to extract available Cu, Zn, As, and Cd in soils of Zhengzhou suburbs, where the mean values varied from 0.129 to 6.881 mg/kg. The bioavailability of different heavy metals in the soil varies greatly, with Cd having the highest activity in the survey region. Significant (p < 0.01) positive relationships were observed between the available state and the total amount of all the heavy metals. The assessment of health risks associated with heavy metals indicated that there was no risk for chronic non-carcinogenic effects. Even though the total amount of metal elements in suburban soil of Zhengzhou is 1.6% with high carcinogenic risk, the risk of available elements is still within the acceptable range, which verified that the risk grade obtained by the total amount is higher than the actual risk.

An anaerobic bacterium host system for heterologous expression of natural product biosynthetic gene clusters.

Anaerobic bacteria represent an overlooked rich source of biological and chemical diversity. Due to the challenge of cultivation and genetic intractability, assessing the capability of their biosynthetic gene clusters (BGCs) for secondary metabolite production requires an efficient heterologous expression system. However, this kind of host system is still unavailable. Here, we use the facultative anaerobe Streptococcus mutans UA159 as a heterologous host for the expression of BGCs from anaerobic bacteria. A natural competence based large DNA fragment cloning (NabLC) technique was developed, which can move DNA fragments up to 40-kb directly and integrate a 73.7-kb BGC to the genome of S. mutans UA159 via three rounds of NabLC cloning. Using this system, we identify an anti-infiltration compound, mutanocyclin, from undefined BGCs from human oral bacteria. We anticipate this host system will be useful for heterologous expression of BGCs from anaerobic bacteria.

d-Sedoheptulose-7-phosphate is a common precursor for the heptoses of septacidin and hygromycin B.

Seven-carbon-chain-containing sugars exist in several groups of important bacterial natural products. Septacidin represents a group of l-heptopyranoses containing nucleoside antibiotics with antitumor, antifungal, and pain-relief activities. Hygromycin B, an aminoglycoside anthelmintic agent used in swine and poultry farming, represents a group of d-heptopyranoses-containing antibiotics. To date, very little is known about the biosynthesis of these compounds. Here we sequenced the genome of the septacidin producer and identified the septacidin gene cluster by heterologous expression. After determining the boundaries of the septacidin gene cluster, we studied septacidin biosynthesis by in vivo and in vitro experiments and discovered that SepB, SepL, and SepC can convert d-sedoheptulose-7-phosphate (S-7-P) to ADP-l-glycero-ß-d-manno-heptose, exemplifying the involvement of ADP-sugar in microbial natural product biosynthesis. Interestingly, septacidin, a secondary metabolite from a gram-positive bacterium, shares the same ADP-heptose biosynthesis pathway with the gram-negative bacterium LPS. In addition, two acyltransferase-encoding genes sepD and sepH, were proposed to be involved in septacidin side-chain formation according to the intermediates accumulated in their mutants. In hygromycin B biosynthesis, an isomerase HygP can recognize S-7-P and convert it to ADP-d-glycero-ß-d-altro-heptose together with GmhA and HldE, two enzymes from the Escherichia coli LPS heptose biosynthetic pathway, suggesting that the d-heptopyranose moiety of hygromycin B is also derived from S-7-P. Unlike the other S-7-P isomerases, HygP catalyzes consecutive isomerizations and controls the stereochemistry of both C2 and C3 positions.

Overexpression of metK shows different effects on avermectin production in various Streptomyces avermitilis strains.

The S-adenosylmethionine synthetase gene (metK) from Streptomyces avermitilis was cloned into multi-copy vector pIJ653 and integrative vector pSET152 yielding two metK expression plasmids pYJ02 and pYJ03, respectively. When wild-type strain ATCC31267 was transformed with these two plasmids, avermectin production was increased about 2.0-fold and 5.5-fold, respectively. The introduction of integrative expression plasmid pYJ03 into the engineered strain GB-165, which produces only avermectin B, promoted the production of avermectin approximately 2.0-fold. However, introduction of pYJ02 did not influence avermectin accumulation in GB-165. Moreover, transformation of the avermectin-overproducing industry strain 76-05 with these two plasmids did not stimulate avermectin production. These results showed that there were different effects of metK expression levels on avermectin production in various S. avermitilis strains. Additionally, the transcript levels of metK, aveR (the avermectin pathway-specific regulatory gene) and aveA1 (one avermectin biosynthesis gene) meet the expectation of fermentation levels of avermectin in wild-type strain and its recombinant strains. The gene expression levels of metK, aveR and aveA1 in GB-165 and 76-05 were much higher then those in wild-type strain, which probably limited the increasement of avermectin by overexpression of metK.